College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, People's Republic of China.
Parasitol Res. 2012 Nov;111(5):2157-63. doi: 10.1007/s00436-012-3064-x. Epub 2012 Aug 11.
A high-resolution melting (HRM) assay was applied to distinguish between Giardia duodenalis assemblages A and B from human and dog feces based on the triosephosphate isomerase gene (tpi). The genomic DNAs were selected from assemblages A (WB) and B (GS) as reference and plasmids were constructed. The reference plasmids and genomic DNAs from 15 Giardia-positive samples were analyzed by HRM assay. This was followed by separate real-time PCR assays specific for assemblages A and B using EvaGreen (EG) to identify PCR products by melting-point analysis. Our results indicate that PCR with HRM in a one-step closed-tube method is a reliable diagnostic method for G. duodenalis zoonotic assemblage identification and more rapid than restriction length polymorphism analysis and direct sequence analysis, HRM is specific, sensitive, reproducible, and rapid. This study is the first use of EG dye for Giardia genotyping. This assay is a promising approach to determine the presence and genotype of Giardia based on a highly variable gene.
高分辨率熔解（HRM）分析应用于根据三磷酸甘油醛异构酶基因（tpi）区分人源和犬源贾第虫 A、B 两型。选择贾第虫 A 型（WB）和 B 型（GS）的基因组 DNA 作为参考，并构建质粒。用 HRM 分析对 15 份贾第虫阳性样本的参考质粒和基因组 DNA 进行分析。然后使用 EvaGreen（EG）对特定于 A、B 两型的实时 PCR 进行单独分析，通过熔点分析鉴定 PCR 产物。我们的结果表明，一步法封闭管内 HRM-PCR 是一种可靠的贾第虫动物源两型鉴定的诊断方法，比限制性长度多态性分析和直接序列分析更快，HRM 具有特异性、敏感性、可重复性和快速性。本研究首次将 EG 染料用于贾第虫基因分型。该方法是一种基于高度变异基因确定贾第虫存在和基因型的有前途的方法。
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